We have evidence that certain hypothalamic hypophysiotropic substances (CRF, PRF, TRF) may exist as large proteins. In the present proposal, we will extend our studies on "big" and "little" CRF to other hypothalamic hypophysiotropic hormones (TRF, PRF, LH-RH) to see whether high molecular weight formms of most or all hypothalamic hypophysiotropic hormones can be identified. RIA measurement of the appropriate adenohypophysial hormones secreted by monolayer cultures of rat adenohypophysial cells or direct assessment of hypothalamic hormones by RIA will be employed to determine the hypophysiotrophic activities of column fractions of bovine hypothalamic or pituitary stalk extracts. To clarify its chemical characteristics big CRF will be treated with various protein-denaturing substances, thiol compounds, or proteolytic enzymes to see whether such treatments yield "little" CRF. The precise molecular size of "big" CRF will be estimated with agarose-gel and SDS polyacrylamide gel electrophoresis (SDS-PAGE), and the molecular size of "little" CRF with SDS-PAGE. Concanavalin-A (Con-A) agarose will be used to determine if CRF molecule(s) have a glycoprotein component. Combination of gel filtration, ion-exchange chromatography, Con-A, SDS-PAGE, and isoelectric focusing will be used for further purification of "big" and "little" CRFs. The molecular form of CRF present in the secretory granules or synaptosomes will be studied by sucrose density gradient centrifugation. If high molecular weight releasing hormones other than CRF can be isolated the same approach described above for CRF will be applied to clarify their nature. These studies should provide important new information relative to how hypothalamic hypophysiotropic hormones are synthesized and secreted from the hypothalamus, and expedite isolation and characterization of these hormones.